波尔山羊精子体外获能及获能前后超微结构的变化
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摘要
本文就国内新引进的波尔山羊精子体外获能及获能前后超微结构的变化进行
研究。获能处理前先进行活精子分离，然后按实验之需，用含有肝素的培养液获
能处理，获能结束后用溶血磷脂酰胆碱（LC）诱导顶体反应，TG染色法测得有顶
体反应的活精子作为评价精子获能的指标。肝素添加剂量设5个水平，即0μg/ml、
5μg/ml、10μg/ml、15μg/ml、20μg/ml；处理时间分4h、3h、2h、1h、15min5个
水平；LC处理时间设0min、3min、6min、9min、12min、15min、20min7个水平。
电镜样品用常规方法固定、脱水、包埋、聚合，LKB-2088V切片机切片，铀铅片染，
JEM-100XII观察，拍照。
结果表明：三种培养液中，从获能效果、精子活力和活精子百分率来考虑，S
培养液效果最好，4h的顶体反应率达60.88％，与D、B培养液的获能效果相比，差
异极显著（P＜0.01），获能4h和6h的效果差异不显著（P＞0.05）；肝素的添加
剂量以15μg/ml和20μg/ml的效果最好，与其它处理组的效果相比差异显著
（P＜0.05），但这两个水平间的效果差异不显著（P＞0.05）；LC诱导顶体反应的
时间在15min时效果极显著高于15min以下处理组的效果（P＜0.01）。羊精子的
超微结构与其它哺乳动物的类似，分头、颈、尾三部分。头部主要由细胞核构成，
含有遗传物质；颈部脆弱，尾部易从此脱落；尾部由中段、主段和末段组成。精
子获能后，顶体质膜膨胀、断裂和丢失；顶体外膜囊泡化，进而顶体内膜裸露。
IN VITRO CAPACITAT ION OF BOER SPERM AND ITS ULTRU?STRACTURE
BEFORE OR AFTER CAPACITAT ION
Abstract : The objectives of this study were to select the excellent culture media lii
vitro capacitafion and observe the fine structure of Boer sperm before or after capacitatiom
Three cultwe media used are 5, D and B. Separate the motile spennatozoa before
capacitation, and lhen exposure capacitated sperm to lysophosphatidyl-choline(LC) to
induce acmsome reaction. Capacitation was evaluated by determining the number of
motile aciosome-reacted sperm using TG staining. Sperm were incubated 4h by S media
of0 g/ml 5p.g/mU. 10 jig/mb 15 jig/mi-. 2Ojig/mlheparin,andheparinwasaddedat
0h 1W. 2W. 3W. 3.75hafterthe start of incubation. Atthe end of incubation, Oor3Op.gIml
LC was added, and the percentage of sperm acrosome-reacted was determined by TG
staining after a thither 1 5mm incubation. Obtain the sperm capacitated or non-capacitated
and process according to routine procedures for TEM.
Results indicated S media was the best culture media allowing for its effects on
capacitation, motility and viability of sperm. The percentage of acrosome-reacted sperm
at 4h was significantly different from other t media (P < 0.01) . Supplements of
1 5jig-l or 20 jig/mI heparin had apparent effect on acrosome reaction of sperm (P <
0.05) . But there was no apparent difference between these two treatments (P >0.05).
Induce capacitated sperm with LC for a further 15mm and obtain the results of
acmsome-reacted sperm which was significantly different from any other treatments (P <
0.01) . But there were difference among individuals. The ultra-stmcture of Boer
spermatozoa was similar to that of other mammals. Eveiy sections of sperm had its ox
characteristics. The great morphological changes had happened after capacitation. and that
maybe have significant importance in fertilization.
研究。获能处理前先进行活精子分离，然后按实验之需，用含有肝素的培养液获
能处理，获能结束后用溶血磷脂酰胆碱（LC）诱导顶体反应，TG染色法测得有顶
体反应的活精子作为评价精子获能的指标。肝素添加剂量设5个水平，即0μg/ml、
5μg/ml、10μg/ml、15μg/ml、20μg/ml；处理时间分4h、3h、2h、1h、15min5个
水平；LC处理时间设0min、3min、6min、9min、12min、15min、20min7个水平。
电镜样品用常规方法固定、脱水、包埋、聚合，LKB-2088V切片机切片，铀铅片染，
JEM-100XII观察，拍照。
结果表明：三种培养液中，从获能效果、精子活力和活精子百分率来考虑，S
培养液效果最好，4h的顶体反应率达60.88％，与D、B培养液的获能效果相比，差
异极显著（P＜0.01），获能4h和6h的效果差异不显著（P＞0.05）；肝素的添加
剂量以15μg/ml和20μg/ml的效果最好，与其它处理组的效果相比差异显著
（P＜0.05），但这两个水平间的效果差异不显著（P＞0.05）；LC诱导顶体反应的
时间在15min时效果极显著高于15min以下处理组的效果（P＜0.01）。羊精子的
超微结构与其它哺乳动物的类似，分头、颈、尾三部分。头部主要由细胞核构成，
含有遗传物质；颈部脆弱，尾部易从此脱落；尾部由中段、主段和末段组成。精
子获能后，顶体质膜膨胀、断裂和丢失；顶体外膜囊泡化，进而顶体内膜裸露。
IN VITRO CAPACITAT ION OF BOER SPERM AND ITS ULTRU?STRACTURE
BEFORE OR AFTER CAPACITAT ION
Abstract : The objectives of this study were to select the excellent culture media lii
vitro capacitafion and observe the fine structure of Boer sperm before or after capacitatiom
Three cultwe media used are 5, D and B. Separate the motile spennatozoa before
capacitation, and lhen exposure capacitated sperm to lysophosphatidyl-choline(LC) to
induce acmsome reaction. Capacitation was evaluated by determining the number of
motile aciosome-reacted sperm using TG staining. Sperm were incubated 4h by S media
of0 g/ml 5p.g/mU. 10 jig/mb 15 jig/mi-. 2Ojig/mlheparin,andheparinwasaddedat
0h 1W. 2W. 3W. 3.75hafterthe start of incubation. Atthe end of incubation, Oor3Op.gIml
LC was added, and the percentage of sperm acrosome-reacted was determined by TG
staining after a thither 1 5mm incubation. Obtain the sperm capacitated or non-capacitated
and process according to routine procedures for TEM.
Results indicated S media was the best culture media allowing for its effects on
capacitation, motility and viability of sperm. The percentage of acrosome-reacted sperm
at 4h was significantly different from other t media (P < 0.01) . Supplements of
1 5jig-l or 20 jig/mI heparin had apparent effect on acrosome reaction of sperm (P <
0.05) . But there was no apparent difference between these two treatments (P >0.05).
Induce capacitated sperm with LC for a further 15mm and obtain the results of
acmsome-reacted sperm which was significantly different from any other treatments (P <
0.01) . But there were difference among individuals. The ultra-stmcture of Boer
spermatozoa was similar to that of other mammals. Eveiy sections of sperm had its ox
characteristics. The great morphological changes had happened after capacitation. and that
maybe have significant importance in fertilization.
引文
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2．袁玉英等．1998b．r-氨基丁酸诱发人精子顶体反应及其受精能力．生理学报 50(3)：326～332
3．石其贤等．1988．鞘磷脂酶加速体外豚鼠精子顶体反应 科学通报 34(23)：1827～1830
4．李明文等．1996b．豚鼠精子信号通路的研究中国科学(C辑)26：363～368
5．石其贤，贾振宇．1995．哺乳动物精子顶体反应及其信息传导生殖与避孕 15(2)：83～87
6．袁玉英等．1998a．卵丘细胞及胞外基质诱发仓鼠精子顶体反应解剖学报 29(4)：404～409
7．贾振宇等．1997．孕酮诱发豚鼠精子顶体反应过程中的C2~(2+)内流通道．生理学报 49(3)：349～353
8．中国科学院新疆化学研究所电镜组．1980．畜牧兽医学报11(1)
9．石其贤等．1991．羊精子体外获能．动物学报 37(1)：76～83
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11．周占祥．1994．牛精子表面糖复合物的分布及洗涤的影响．解剖学报 25(2)：177～180
12．周红等．1992．精子与卵细胞膜上糖复合物在生殖过程中的作用．生殖与避孕 12(1)：3
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16．李明文等．1995．大熊猫精子获能和顶体反应过程中钙分布规律的研究．动物学报 41(4)：420～424
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2．袁玉英等．1998b．r-氨基丁酸诱发人精子顶体反应及其受精能力．生理学报 50(3)：326～332
3．石其贤等．1988．鞘磷脂酶加速体外豚鼠精子顶体反应 科学通报 34(23)：1827～1830
4．李明文等．1996b．豚鼠精子信号通路的研究中国科学(C辑)26：363～368
5．石其贤，贾振宇．1995．哺乳动物精子顶体反应及其信息传导生殖与避孕 15(2)：83～87
6．袁玉英等．1998a．卵丘细胞及胞外基质诱发仓鼠精子顶体反应解剖学报 29(4)：404～409
7．贾振宇等．1997．孕酮诱发豚鼠精子顶体反应过程中的C2~(2+)内流通道．生理学报 49(3)：349～353
8．中国科学院新疆化学研究所电镜组．1980．畜牧兽医学报11(1)
9．石其贤等．1991．羊精子体外获能．动物学报 37(1)：76～83
10．秦鹏春，石惠艺．1997．猪精子冷冻损伤的研究．国外畜牧学(猪与禽) 33～36
11．周占祥．1994．牛精子表面糖复合物的分布及洗涤的影响．解剖学报 25(2)：177～180
12．周红等．1992．精子与卵细胞膜上糖复合物在生殖过程中的作用．生殖与避孕 12(1)：3
13．朱正美，程腊梅．1992．细胞表面糖复合物及妊娠．生殖与避孕 12(2)：3
14．周占祥，邓泽沛．1994．羊精子成熟和获能过程中表面的凝集素标记变化．动物学报25(3)：277～280
15．杨增明和陈大元．1992．豚鼠精子在发生及顶体反应过程中细胞内Ca~(2+)的定位研究．动物学报38(4)：425～427
16．李明文等．1995．大熊猫精子获能和顶体反应过程中钙分布规律的研究．动物学报 41(4)：420～424
17．李明文等．1995．猕猴精子获能和顶体反应过程中Ca~(2+)和Ca~(2+)-ATPase定位及咖啡因和dbcAMP对顶体反应的影响．解剖学报 26(2)：146～151
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